Artificial red blood cells

ABSTRACT

An artificial red blood cells are disclosed comprising a thin film encapsulating a core, the core comprising hemoglobin, wherein the film comprises a plurality of layers of polypeptides, opposing layers comprising alternating oppositely charged polypeptides, and wherein at least half of the amino acid residues in the oppositely charged polypeptides have the same charge. In one embodiment, a first layer comprises a first layer polypeptide comprising one or more first amino acid sequence motifs, wherein the one or more amino acid sequence motifs consist of n amino acids, at least x of which are positively charged, or at least x of which are negatively charged, wherein x is greater than or equal to approximately one-half of n.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of U.S. Nonprovisional application Ser.No. 10/652,364 filed Aug. 29, 2003, which is incorporated by referenceherein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the fabrication of ultrathinmultilayered films on suitable surfaces by electrostatic layer-by-layerself assembly (“ELBL”). More specifically, the present invention relatesto a method for designing polypeptides for the nanofabrication of thinfilms, coatings, and microcapsules by ELBL for applications inbiomedicine and other fields.

2. Description of Related Art

ELBL is an established technique in which ultrathin films are assembledby alternating the adsorption of oppositely-charged polyelectrolytes.The process is based on the reversal of the surface charge of the filmafter the deposition of each layer. FIG. 1 shows a schematic diagram ofthe general ELBL process: films of oppositely charged polyions (cationicpolyions 10 and anionic polyions 11) are assembled in successive layerson a negatively-charged planar surface 12; the surface charge isreversed after the deposition of each layer. This process is repeateduntil a film of desired thickness is formed. The physical basis ofassociation is electrostatics—gravitation and nuclear forces playeffectively no role. Because of the generality and relative simplicityof the process, ELBL allows for the deposition of many different typesof materials onto many different types of surface. There is, therefore,a vast number of possible useful combinations of materials and surfaces.For a general discussion of ELBL, including its history, see Yuri Lvov,“Electrostatic Layer-by-Layer Assembly of Proteins and Polyions” inProtein Architecture: Interfacial Molecular Assembly and ImmobilizationBiotechnology, Y. Lvov & H. Mohwald eds. (New York: Marcel Dekker,1999), pp. 125-167, which is incorporated herein by reference in itsentirety.

ELBL has recently become a focus area in the field of nanotechnologybecause it can be used to fabricate films substantially less than 1micron in thickness. Moreover, ELBL permits exceptional control over thefilm fabrication process, enabling the use of nanoscale materials andpermitting nanoscale structural modifications. Because each layer has athickness on the order of a few nanometers or less, depending on thetype of material used and the specific adsorption process, multilayerassemblies of precisely repeatable thickness can be formed.

A number of synthetic polyelectrolytes have been employed in ELBLapplications, including sodium poly(styrene sulfonate) (“PSS”),poly(allylamine hydrochloride) (“PAH”), poly(diallyldimethylammoniumchloride) (“PDDA”), poly(acrylamide-co-diallyldimethylammoniumchloride), poly(ethyleneimine) (“PEI”), poly(acrylic acid) (“PAA”),poly(anetholesulfonic acid), poly(vinyl sulfate) (“PVS”), andpoly(vinylsulfonic acid). Such materials, however, are not generallyuseful for biomedical applications because they are antigenic or toxic.

Proteins, being polymers with side chains having ionizable groups, canbe used in ELBL for various applications, including biomedical ones.Examples of proteins that have been used in ELBL include cytochrome c,hen egg white lysozyme, immunoglobulin G, myoglobin, hemoglobin, andserum albumin (ibid.). There are, however, difficulties with usingproteins for this purpose. These include limited control over multilayerstructure (because the surface of the protein is highly irregular andproteins will not ordinarily adsorb on a surface in a regular pattern),restrictions on pH due to the pH-dependence of protein solubility andstructural stability, lack of biocompatibility when using exogenousproteins, and the cost of scaling up production if the gene has not beencloned; unless the protein were identical in a readily available source,e.g. a cow, the protein would have to be obtained from the organism inwhich it was intended for use, making the cost of large-scale productionof the protein prohibitive.

By contrast polypeptides, which are generally smaller and less complexthan proteins, constitute an excellent class of material for ELBLassembly, and polypeptide film structures formed by ELBL will be usefulin a broad range of applications. The present invention provides amethod for designing polypeptides for the nanofabrication of thin films,coatings, and microcapsules by ELBL. Polypeptides designed using themethod of the present invention should exhibit several usefulproperties, including, without limitation, completely determined primarystructure, minimal secondary structure in aqueous solution,monodispersity, completely controlled net charge per unit length,ability to form cross-links on demand, ability to reverse cross-linkformation, ability to form more organized thin films than is possiblewith proteins, and relatively inexpensive large-scale production cost(assuming gene design, synthesis, cloning, and host expression in E.coli or yeast, or peptide synthesis).

Polypeptides designed using the method of the present invention havebeen shown useful for ELBL of thin film structures with targeted orpossible applications in biomedical technology, food technology, andenvironmental technology. Such polypeptides could be used, for example,to fabricate artificial red blood cells, drug delivery devices, andantimicrobial films.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a novel method for identifying “sequencemotifs” of a defined length and net charge at neutral pH in amino acidsequence information for use in ELBL, and recording a desired number ofthe motifs. The method comprises the steps of: (a) Obtaining an aminoacid sequence for a peptide or a protein from a particular organism; (b)Locating a starter amino acid in the amino acid sequence; (c) Examiningthe starter amino acid and the following n amino acids to determine thenumber of charged amino acids having a polarity opposite the certainpolarity; (d) If the number of the charged amino acids having a polarityopposite the certain polarity is one or more, continuing the method atstep g; (e) Examining the starter amino acid and the following n aminoacids to determine the number of charged amino acids having the certainpolarity; (f) If the number of charged amino acids having the certainpolarity is equal to or greater than x, recording the amino acidsequence motif consisting of the starter amino acid and the following namino acids; (g) Locating another starter amino acid in the amino acidsequence; and (h) Repeating the method beginning at step c until thedesired number of amino acid sequence motifs have been identified or allof the amino acids in the amino acid sequence have been used as thestarter amino acid in step c; wherein x is greater than or equal toapproximately one-half of n.

The present invention also provides a novel method for designing apolypeptide for use in ELBL, comprising the steps of: (a) Identifyingand recording one or more amino acid sequence motifs having a net chargeof a certain polarity using the steps mentioned in the precedingparagraph and (b) Joining a plurality of said recorded amino acidsequence motifs to form a polypeptide.

The present invention also provides a novel method for designing apolypeptide for use in ELBL comprising the following steps: (a)Designing de novo a plurality of amino acid sequence motifs, whereinsaid amino acid sequence motifs consist of n amino acids, at least x ofwhich are positively charged and none is negatively charged, or at leastx of which are negatively charged and none is positively charged,wherein x is greater than or equal to approximately one-half of n; and(b) Joining said plurality of said amino acid sequence motifs. The aminoacid sequence motifs can comprise the 20 usual amino acids ornon-natural amino acids, and the amino acids can be either left-handed(L-amino acids) or right handed (D-amino acids).

The present invention also provides a thin film, the film comprising aplurality of layers of polypeptides, the layers of polypeptides havingalternating charges, wherein the polypeptides comprise at least oneamino acid sequence motif consisting of n amino acids, at least x ofwhich are positively charged and none is negatively charged, or at leastx of which are negatively charged and none is positively charged,wherein x is greater than or equal to approximately one-half of n. Themotifs in these polypeptides may be selected using either of the methodsdescribed above.

The present invention also provides a novel process for using cysteineand other sulphydryl-containing amino acid types to “lock” and “unlock”the layers of polypeptide ELBL films. This process enables the films toremain stable at extremes of pH, giving greater control over themechanical stability and diffusive properties of films nanofabricatedfrom designed polypeptides and increasing their utility in a broad rangeof applications.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a schematic diagram of the general ELBL process.

FIG. 2 is a graph of the cumulative secondary structure propensities ofthe amino acid sequence motifs identified in human amino acid sequenceinformation using the method of the present invention, compared with thedistribution of structure propensities of 105 random amino acidsequences.

FIG. 3(a) shows adsorption data as monitored by the quartz crystalmicrobalance technique (“QCM”) for a combination of amino acid sequencesdesigned according to the present invention.

FIG. 3(b) shows a comparison of adsorption data as monitored by QCM fordifferent combinations of amino acid sequences designed according to thepresent invention.

FIG. 3(c) shows a graph of adsorbed mass in nanograms versus layernumber for amino acid sequences designed and fabricated according to thepresent invention.

FIG. 4(a) illustrates intra-layer disulfide bonds according to thecysteine locking method of the present invention.

FIG. 4(b) illustrates inter-layer disulfide bonds according to thecysteine locking method of the present invention.

FIG. 4(c) illustrates the oxidation and reduction of disulfide bonds inmicrocapsules fabricated from polypeptides designed according to themethod of the present invention.

FIG. 5 is a schematic of the selection process of the present inventionused to identify in existing amino acid sequence information amino acidsequence motifs having suitable electrostatic properties for ELBL.

FIG. 6 shows the number of non-redundant sequence motifs identified inavailable human amino acid sequence data.

FIG. 7 shows the ELBL adsorption of poly-L-glutamate and poly-L-lysinefrom an aqueous medium as a function of ionic strength.

FIG. 8 shows the adsorption of polypeptides designed according to themethod of the present invention for experiments to probe the effect ofdisulfide bond formation.

FIG. 9 shows the percentage of material remaining during thin filmdisassembly at acidic pH as discussed with reference to FIG. 8.

FIG. 10 shows the percentage of material lost during the acidic pHdisassembly step of an experiment involving de novo-designedpolypeptides containing cysteine.

FIG. 11(a) illustrates the role of solution structure of peptides onfilm assembly, showing how the assembly behavior of poly-L-glutamate andpoly-L-lysine depends on pH. QCM resonant frequency is plotted againstadsorption layer. The average molecular mass of poly-L-glutamate was84,600 Da, while that of poly-L-lysine was 84,000 Da. The numbers referto pH values. E=Glu, K=Lys. The peptide concentration used for assemblywas 2 mg/mL.

FIG. 11(b) illustrates the role of solution structure of peptides onfilm assembly, showing how the solution structure of poly-L-glutamateand poly-L-lysine depends on pH. Mean molar residue ellipticity isplotted as a function of pH. The peptide concentration was 0.05 mg/mL.

FIG. 12 shows adsorption data for polyelectrolytes of different lengths,illustrating that long polyelectrolytes adsorb better than short ones.

DETAILED DESCRIPTION OF THE INVENTION

Explanations of Terms

For convenience in the ensuing description, the following explanationsof terms are adopted. However, these explanations are intended to beexemplary only. They are not intended to limit the terms as they aredescribed or referred to throughout the specification. Rather, theseexplanations are meant to include any additional aspects and/or examplesof the terms as described and claimed herein.

As used herein, “biocompatibility” means causing no adverse healtheffect upon ingestion, contact with the skin, or introduction to thebloodstream.

As used herein, “immune response” means the response of the human immunesystem to the presence of a substance in the bloodstream. An immuneresponse can be characterized in a number of ways, for example, by anincrease in the bloodstream of the number of antibodies that recognize acertain antigen. (Antibodies are proteins made by the immune system, andan antigen is an entity that generates an immune response.) The humanbody fights infection and inhibits reinfection by increasing the numberof antibodies in the bloodstream. The specific immune response dependssomewhat on the individual, though general patterns of response are thenorm.

As used herein, “epitope” means the structure of a protein that isrecognized by an antibody. Ordinarily an epitope will be on the surfaceof a protein. A “continuous epitope” is one that involves several aminoacids in a row, not one that involves amino acid residues that happen tobe in contact in a folded protein.

As used herein, “sequence motif” and “motif” mean an amino acid sequenceof a given number of residues identified using the method of the currentinvention. In a preferred embodiment, the number of residues is 7.

As used herein, “amino acid sequence” and “sequence” mean any length ofpolypeptide chain that is at least two amino residues long.

As used herein, “residue” means an amino acid in a polymer; it is theresidue of the amino acid monomer from which the polymer was formed.Polypeptide synthesis involves dehydration—a single water molecule is“lost” on addition of the amino acid to a polypeptide chain.

As used herein, “designed polypeptide” means a polypeptide designedusing the method of the present invention, and the terms “peptide” and“polypeptide” are used interchangeably.

As used herein, “primary structure” means the linear sequence of aminoacids in a polypeptide chain, and “secondary structure” means the moreor less regular types of structure stabilized by non-covalentinteractions, usually hydrogen bonds—examples include α-helix, β-sheet,and β-turn.

As used herein, “amino acid” is not limited to the 20 naturallyoccurring amino acids; the term also refers to D-amino acids, L-aminoacids, and non-natural amino acids, as the context permits.

As used herein, “non-natural amino acids” means amino acids other thanthe 20 naturally occurring ones.

The following three-letter abbreviations are used herein for the 20usual amino acids: Ala = alanine Cys = cysteine Asp = aspartic acid Glu= glutamic acid Phe = phenylalanine Gly = glycine His = histidine Ile =isoleucine Lys = lysine Leu = leucine Met = methionine Asn = asparaginePro = proline Gln = glutamine Arg = arginine Ser = serine Thr =threonine Val = valine Trp = tryptophan Tyr = tyrosine

A. Description of the Invention

The present invention provides a method for designing polypeptides forthe nanofabrication by ELBL of thin films, coatings, and microcapsulesfor applications in biomedicine and other fields. The method involves 5primary design concerns: (1) the electrostatic properties of thepolypeptides; (2) the physical structure of the polypeptides; (3) thephysical stability of the films formed from the polypeptides; (4) thebiocompatibility of the polypeptides and films; and (5) the bioactivityof the polypeptides and films. The first design concern, electrostatics,is perhaps the most important because it is the basis of ELBL. Withoutsuitable charge properties, a polypeptide will not be soluble in aqueoussolution and cannot be used for the ELBL nanofabrication of films. Wehave devised a novel process for identifying in amino acid sequenceinformation amino acid sequence motifs having electrostatic propertiessuitable for ELBL.

The secondary structure of the polypeptides used for ELBL is alsoimportant, because the physical properties of the film, including itsstability, will depend on how the solution structure of the peptidetranslates into its structure in the film. FIG. 11 illustrates how thesolution structure of certain polypeptides correlates with filmassembly. Panel (a) shows how the assembly behavior of poly-L-glutamateand poly-L-lysine depends on pH. It is clear that the α-helixconformation correlates with a greater extent of deposited material thanthe β-sheet conformation. The precise molecular interpretation of thisbehavior remains to be elucidated. Panel (b) shows how the solutionstructure of these peptides depends on pH. At pH 4.2 poly-L-glutamate islargely α-helical, as is poly-L-lysine at pH 10.5. Both polypeptides arein a largely unstructured coil-like conformation at pH 7.3.

The remaining concerns relate to the applications of the polypeptidefilms. In practicing the invention, more or less weight will be placedon these other concerns depending on the design requirements of aparticular application.

By using the selection process of the present invention to identify inamino acid sequence information amino acid sequence motifs havingsuitable charge characteristics, and using the other design concerns toselect particular motifs, one can design polypeptides suitable for theELBL fabrication of nano-organized films for applications in biomedicineand other fields. Alternatively, one can use the method of the presentinvention to design polypeptides de novo for use in ELBL. The approachto de novo design is essentially the same as identifying motifs inexisting amino acid sequence information, except that each residue in anamino acid sequence motif is selected by the practitioner rather than anentire motif being identified in the genomic or proteomic information ofa specific organism. It must be emphasized that the fundamentalpolypeptide design principles adduced in the present invention areindependent of whether the amino acids involved are the 20naturally-occurring ones, non-natural amino acids, or some novelcombination of these, in the case of de novo polypeptide design.Further, both D-amino acids and L-amino acids could be used.

The design concerns of the present invention are discussed in moredetail below.

1. Electrostatics

We have devised a novel process for identifying in amino acid sequenceinformation amino acid sequence motifs having electrostatic propertiessuitable for ELBL. Using this process, we have identified 88,315non-redundant amino acid sequence motifs in human proteome data—thetranslation of the portion of the genome that encodes all known proteinsin the human body. This information is publicly available at theNational Center for Biotechnology Information's (“NCBI”) Web site:<http://www.ncbi.nlm.nih.gov>, among other places. Such information isconstantly being updated as the human genome is further analyzed. As theamount of such information increases, the number of amino acid sequencemotifs that could be identified in human sequence information by theselection process of the present invention as having suitableelectrostatic properties for ELBL will also increase. The same is truefor any organism. Accepted biochemical and physics principles, as wellas the experimental results described below, indicate that theidentified sequence motifs will be useful for the design of polypeptidesfor the nanofabrication of ELBL structures.

The key selection criterion is the average charge per unit length atneutral pH (pH 7, close to the pH of human blood). In addition, thereare several structural preferences. First, it is preferred that eachamino acid sequence motif consist of only 7 residues.

a. Total Number of Residues in the Motif

The motif length of 7 was chosen in an effort to optimizebiocompatibility, physical structure, and the number of non-redundantsequence motifs in available amino acid sequence data.

As discussed below, it is preferred that at least half of the amino acidresidues in each sequence motif be charged. Moreover, it is preferredthat all of the charged residues in each motif be of the same charge.These requirements ensure that each motif will be sufficiently solublein aqueous solvent and have sufficient charge at neutral pH to be usefulfor ELBL. Because only a relatively small percentage of amino acid typesare charged, as the length of a given amino acid sequence increases, theodds decrease that the sequence will have a sufficient percentage ofappropriately charged amino acids for ELBL. 4 charged amino acids is thepreferred minimum for a motif size of 7, because fewer than 4 chargesyields substantially decreased peptide solubility and decreased controlover ELBL.

Regarding biocompatibility (discussed further below), each identifiedsequence motif is long enough at 7 residues to constitute a continuousepitope (relevant to the possible immune response of an organism intowhich a designed peptide might be introduced), but not so long as tocorrespond substantially to residues both on the surface of a proteinand in its interior; the charge requirements help to ensure that thesequence motif occurs on the surface of the folded protein; a chargedresidue cannot be formed in the core of a folded protein. By contrast, avery short motif could appear to the body to be a random sequence, orone not specifically “self,” and therefore elicit an immune response.Although the ideal length of a peptide for generating antibodies is apoint of some dispute, most peptide antigens range in length from 12 to16 residues. Peptides that are 9 residues or shorter can be effectiveantigens; peptides longer than 12 to 16 amino acids may contain multipleepitopes (Angeletti, R. H. (1999) Design of Useful Peptide Antigens, J,Biomol. Tech. 10:2-10, which is hereby incorporated by reference in itsentirety). Thus, to minimize antigenicity one would prefer a peptideshorter than 12 and, better yet, shorter than 9 residues.

The preferred motifs should not be too long for another reason: tominimize secondary structure formation. Secondary structure decreasescontrol of the physical structure of the polypeptides (see below) andthe films made from them.

Furthermore, the maximum number of non-redundant motifs is found whenthe number of residues in each motif is 7. FIG. 6 shows the number ofnon-redundant sequence motifs in available human amino acid sequenceinformation. The greatest number of positive motifs is for a 5-residuelength, while the greatest number of negative motifs is for a 7-residuelength. The greatest number of positive and negative motifs is about thesame for 5 and 7. Thus, a motif length of 7 residues would appear tomaximize the number of non-redundant motifs.

For all of the above reasons, 7 residues is the preferred length ofmotif to optimize polypeptide design for ELBL. Nevertheless, it ispossible that in some cases either slightly shorter or slightly longermotifs will work equally as well. For example, motifs 5 or 6 residueslong may be employed, and motifs on the order of 8 to 15 residues inlength could also be useful.

b. Number of Charged Residues

Second, it is preferred that at least 4 positively-charged (basic) aminoacids (Arg, His, or Lys) or at least 4 negatively-charged (acidic) aminoacids (Glu or Asp) are present in each 7-residue motif at neutral pH.Combinations of positive and negative charges are disfavored in aneffort to ensure a sufficiently high charge density at neutral pH. It ispossible, however, that a motif containing both positive and negativeamino acids could be useful for ELBL. For example, a slightly longermotif, say of 9 residues, could have 6 positively charged amino acidsand 1 negatively charged amino acid. It is the balance of charge that isimportant—the overall peptide must be either sufficiently positivelycharged or sufficiently negatively charged at neutral pH. Preferredembodiments of the motifs, however, will contain only Glu or Asp or onlyArg, His, or Lys as the charged amino acids (although other non-chargedamino acids could, and ordinarily do, form part of the motifs), unlessnon-natural amino acids are admitted as acidic or basic amino acids.

FIG. 5 is a flow chart showing the steps involved in the selectionprocess for identifying amino acid sequences having suitableelectrostatic properties. It is assumed that only the 20 usual aminoacids are involved. If searching for negatively-charged motifs, theprocess begins by locating an amino acid in the sequence data. Thisamino acid will be called the “starter amino acid” because it is thestarting point for the analysis of the surrounding amino acids (i.e., itwill begin the motif). Next, the starter amino acid and the following 6residues are examined for occurrences of Arg, His, or Lys. If one ormore Arg, His, or Lys is located in these 7 amino acids, the process isbegun anew at another starter amino acid. If no Arg, His, or Lys isfound, the 7 amino acids are examined to determine the number ofoccurrences of Glu and/or Asp. If there are at least 4 occurrences ofGlu and/or Asp in the 7 residues, the sequence motif is cataloged. Theselection process is essentially the same for positively charged aminoacids, except that Glu and Asp are replaced by Arg, His, and Lys, andArg, His, and Lys are replaced by Glu and Asp, respectively. Obviously,one could also begin the method at the beginning of the amino acidsequence (amino terminus) and proceed to the end (carboxyl terminus),or, alternatively, one could begin at a random point and work throughall of the amino acids in the sequence, randomly or systematically ineither direction. Moreover, one could use the method to identify motifsin sequence information containing non-natural amino acids, for exampleif codes were used for each non-natural amino acid type. In such a case,one would search for non-natural acidic or basic amino acids instead ofGlu and Asp, and Arg, Lys, and His, respectively.

The remaining design concerns, namely, physical structure, physicalstability, biocompatibility, and biofunctionality, deal primarily withthe particular application for which the designed polypeptides will beused. As noted above, more or less weight will be placed on theseconcerns during the design process, depending on the desired peptideproperties for a particular application.

2. Physical Structure

A design concern regarding the amino acid sequence motifs is theirpropensity to form secondary structures, notably α-helix or β-sheet. Wehave sought in several ways to control, notably minimize, secondarystructure formation of designed polypeptides in an aqueous medium inorder to maximize control over thin film layer formation. First, it ispreferred that the sequence motifs be relatively short, because longmotifs are more likely to adopt a stable three-dimensional structure insolution. Second, we place a glycine residue between each motif inpreferred embodiments of the polypeptide designs. Glycine has a very lowα-helix propensity and a very low β-sheet propensity, making itenergetically very unfavorable for a glycine and its neighboring aminoacids to form regular secondary structure in aqueous solution. Prolinehas similar properties in some respects and could be used as analternative to glycine to join motifs. Third, we have sought to minimizethe α-helix and β-sheet propensity of the designed polypeptidesthemselves by focusing on motifs for which the summed α-helix propensityis less than 7.5 and the summed β-sheet propensity is less than 8.(“Summed” propensity means the sum of the α-helix or β-sheetpropensities of all amino acids in a motif.) It is possible, however,that amino acid sequences having a somewhat higher summed α-helixpropensity and/or summed β-sheet propensity would be suitable for ELBLunder some circumstances, as the Gly (or Pro) residues between motifswill play a key role in inhibiting stable secondary structure formationin the designed polypeptide. In fact, it may be desirable in certainapplications for the propensity of a polypeptide to form secondarystructure to be relatively high, as a specific design feature of thinfilm fabrication; the necessary electrostatic charge requirements forELBL must still be met, as discussed above.

In order to be able to select amino acid sequences with desiredsecondary structure propensities, we first calculated the secondarystructure propensities for all 20 amino acids using the method of Chouand Fasman (see P. Chou and G. Fasman Biochemistry 13:211 (1974), whichis incorporated by reference herein in its entirety) using structuralinformation from more than 1,800 high-resolution X-ray crystallographicstructures (1,334 containing α-helices and 1,221 containing β-strands).Structures were selected from the Protein Data Bank (apublicly-accessible repository of protein structures) based on: (a)method of structure determination (X-ray diffraction); (b) resolution(better than 2.0 Å)—“resolution” in this context refers to the minimumsize of a structure one can resolve, as in the Rayleigh criterion; and(c) structural diversity (less than 50% sequence identity between theprotein crystallographic structures used to compute the helix and sheetpropensities of the various amino acids). The rationale was to choosehigh resolution structures determined by the most reliable methodologyand not to bias the propensity calculation by having similar structures.Next, for comparison 100,000 non-redundant random sequences wereproduced using a random number generator in a personal computer. We thencalculated the secondary structure propensities for the 88,315 aminoacid sequences identified using the selection process described in partVII(B)(1) above (59,385 non-redundant basic sequence motifs and 28,930non-redundant acidic sequence motifs). The propensities for the randomsequences were then compared to the propensities of the selectedsequences. FIG. 2 shows the distribution of secondary structureformation propensities in these sequence motifs. The rectangle in FIG. 2highlights the sequence motifs we have identified as least likely toform secondary structure on the basis of secondary structurepropensities.

3. Physical Stability

Another design concern is control of the stability of the polypeptideELBL films. Ionic bonds, hydrogen bonds, van der Waals interactions, andhydrophobic interactions provide some, albeit relatively limited,stability to ELBL films. By contrast, covalent disulfide bonds couldprovide exceptional structural strength. We have devised a novel processfor using cysteine (or some other type of sulfhydryl-containing aminoacid) to “lock” and “unlock” adjacent layers of polypeptide ELBL film.This process enables a polypeptide nanofabricated film to remain stableat extremes of pH, giving greater control over its mechanical stabilityand diffusive properties (for discussions of porosity of multilayerfilms made of non-polypeptide polyelectrolytes, see Caruso, F., Niikura,K., Furlong, N. and Okahata (1997) Langmuir 13:3427 and Caruso, F.,Furlong, N., Ariga, K., Ichinose, I., and Kunitake, T. (1998) Langmuir14:4559, both of which are incorporated herein by reference in theirentireties). Also, the incorporation of cysteine (or some other type ofsulfhydryl-containing amino acid) in a sequence motif of a designedpolypeptide enables the use of relatively short peptides in thin filmfabrication, by virtue of intermolecular disulfide bond formation.Without cysteine, such peptides would not generally yield sufficientlystable films (see FIG. 12, discussed below). Thus, our novel use ofcysteine will obviate the need to produce expensive long versions of thedesigned polypeptides in a substantial percentage of possibleapplications. This will be particularly advantageous in situations wherethe thin film is to be fabricated over material to be encapsulated, forexample a small crystal of a drug, a small spherical hemoglobin crystal,or a solution containing hemoglobin.

For applications in which the physical stability of the films isimportant, amino acid sequence motifs containing cysteine (or some othertype of sulfhydryl-containing amino acid) may be selected from thelibrary of motifs identified using the methods discussed above, ordesigned de novo using the principles described above. Polypeptides canthen be designed and fabricated based on the selected or designed aminoacid sequence motifs. Once the polypeptides have been synthesizedchemically or produced in a host organism, ELBL assembly ofcysteine-containing peptides is done in the presence of a reducingagent, to prevent premature disulfide bond formation. Followingassembly, the reducing agent is removed and an oxidizing agent is added.In the presence of the oxidizing agent disulfide bonds form betweencysteine residues, thereby “locking” together the polypeptide layersthat contain them.

This “locking” method may be further illustrated using the followingspecific example of microcapsule fabrication. First, designedpolypeptides containing cysteine are used to form multilayers by ELBL ona suitably charged spherical surface, normally in aqueous solution atneutral pH and in the presence of dithiothreitol (“DTT”), a reducingagent. Next, DTT is removed by filtration, diffusion, or some othersimilar method known in the art, causing cystine to form from pairs ofcysteine side chains and thereby stabilizing the film. If the peptidemultilayers are constructed on a core particle containing the materialsone wishes to encapsulate, for instance a crystalline material, thefabrication process is complete and the core particle can thereafter bemade to dissolve in the encapsulated environment, for example by achange of pH. If, however, the multilayers are constructed on a “dummy”core particle, the core must be removed. In the case of melamineformaldehyde particles (“MF”), for example, the core is ordinarilydissolved by decreasing the pH—dissolution is acid-catalyzed. Followingdissolution of the core, the pH of solution is adjusted to 4, wherepartial charge on the peptide polyanions makes the microcapsulessemi-permeable (compare Lvov et al. (2001) Nano Letters 1:125, which ishereby incorporated herein in its entirety). Next, 10 mM DTT is added tothe microcapsule solution to reduce cystine to cysteine. Themicrocapsules may then be “loaded” by transferring them to aconcentrated solution of the material to be encapsulated, for example aprotein (ibid.). The protein enters the microcapsules by moving down itsconcentration gradient. The encapsulated protein is “locked in” byremoval of reductant and addition of oxidant, thereby promoting thereformation of disulfide bonds.

A schematic of the cysteine “locking” and “unlocking” method of thepresent invention is shown in FIG. 4. Cysteine can form both intra- andinter-molecular disulfide bonds. Further, disulfide bonds can be formedbetween molecules in the same layer or adjacent layers, depending on thelocation of cysteine-containing peptides in the film. Referring to FIG.4(a), basic polypeptides 2 are linked by disulfide bonds 3 in all layersin which the basic peptides contain cysteine. The acidic peptides of theintervening layer (represented in the figure by a translucent layer 4)do not contain cysteine. However, alternating layers continue to attracteach other electrostatically, if the acidic and basic side chains arecharged at the pH of the surrounding environment. Referring to FIG.4(b), disulfide bonds are shown between layers. Such structures willform when both the acidic and basic polypeptides (i.e., alternatingpolypeptide layers) used for ELBL contain cysteine and the procedureused has been suitable for disulfide bond formation. Referring to FIG.4(c), reduction and oxidation reactions are used to regulate the releaseof encapsulated compounds 5 by breaking and forming disulfide bonds 3,respectively, and thereby regulating the diffusion of particles throughthe capsule wall.

The cysteine “locking” and “unlocking” is a novel way of regulating thestructural integrity and permeability of ELBL films. It is known in theart that glutaraldehyde can be used to cross-link proteins, and thischemical could therefore be used to stabilize polypeptide films.Glutaraldehyde cross-linking, however, is irreversible. In contrast, thecysteine “locking” and “unlocking” method of the present invention isreversible and, therefore, offers better control over structureformation and, importantly, use of the films and capsules that can befabricated using the present invention. Blood is an oxidizingenvironment. Thus, in certain biomedical applications, for exampleartificial red blood cells or drug delivery systems fabricated fromdesigned polypeptides, exposing Cys-crosslinked polypeptide film to theblood or some other oxidizing environment after the formation ofdisulfide bonds is not expected to cause those bonds to be broken.Finally, it should also be noted that applications involving non-naturalamino acids would replace Cys with some other sulfhydryl-containingamino acid type. For example, a sulfhydryl could be added to β-aminoacids such as D,L-β-amino-β-cylohexyl propionic acid;D,L-3-aminobutanoic acid; or 5-(methylthio)-3-aminopentanoic acid (seehttp://www.synthatex.com).

4. Biocompatibility

Biocompatibility is a major design concern in biomedical applications.In such applications, the practitioner of the present invention will aimto identify genomic or proteomic information that will yield “immuneinert” polypeptides, particularly if the fabricated or coated objectwill make contact with circulating blood. For purposes of the presentinvention, it is preferred that the selection process discussed in PartVII(B)(1) above be used to analyze the amino acid sequences of bloodproteins. This will maximize the odds of minimizing the immune responseof an organism.

Computer algorithms exist for predicting the antigenicity of an aminoacid sequence. Such methods, however, are known in the art to besemi-reliable at best. In the present invention, the sequence motifsidentified using the selection method discussed above in Part VII(B)(1)are highly polar. The motifs must, therefore, occur on the surface ofthe native state of the proteins of which they are part of the sequence.The “surface” is that part of a folded protein that is in contact withthe solvent or inaccessible to the solvent solely because of thegranular nature of water. The “interior” is that part of a foldedprotein that is inaccessible to solvent for any other reason. A foldedglobular soluble protein is like an organic crystal, the interior beingas densely packed as in a crystal lattice and the exterior being incontact with the solvent, water. Because of their charge properties, thepolypeptide sequence motifs identified using the method of the presentinvention must occur mostly, if not exclusively, on the surface of aprotein. Thus, all of the sequence motifs identified in human bloodproteins using the selection process of the current invention areeffectively always in contact with the immune system while the proteinis in the blood. This holds for all conformations of the protein thatmight become populated in the bloodstream, including denatured states,because it is highly energetically unfavorable to transfer a charge froman aqueous medium to one of low dielectric (as occurs in a proteininterior). Accepted biochemical principles indicate, therefore, that thepolypeptides designed from blood proteins using the method of thepresent invention will either not illicit an immune response or willelicit a minimal immune response. For the same reasons, polypeptidesdesigned using the method of the present invention should bebiocompatible. All sequence motifs identified from genomic data usingthe selection process of the current invention, not only those in bloodproteins, should be biocompatible, though the extent of immune responseor any other type of biological response may well depend on specificdetails of a sequence motif. (Because the polypeptide sequences on whichthe motifs are based actually occur in the organism for which the filmas been fabricated, this approach will, at least in principle, workequally well for any type of organism. For instance, the approach may beof significant value to veterinary science.) Both immune response andbiocompatibility are important regarding the use of the designedpeptides in biomedical applications, including, without limitation, themanufacture of artificial red blood cells, drug delivery systems, orpolypeptides for fabrication of biocompatible films to coat implants forshort-term or long-term introduction into an organism.

5. Bioactivity

In some applications of polypeptide thin films, coatings, ormicrocapsules, it may be desirable to modify the design of thepolypeptides to include a functional domain for use in some layer of thestructure, often the outermost. A functional domain in this context isan independently thermostable region of a protein that has specificbiofunctionality (e.g. binding phosphotyrosine). It is well known in theart that such biofunctionality may be integrated with otherfunctionalities in a multi-domain protein, as for example in the proteintensin, which encompasses a phosphotyrosine binding domain and a proteintyrosine phosphatase domain. The inclusion of such a domain in adesigned polypeptide could function in a number of ways, includingwithout limitation specific ligand binding, targeting in vivo,biosensing, or biocatalysis.

B. Uses for Polypeptides Designed Using the Method of the PresentInvention

As noted above, polypeptides of suitable design are excellent materialsfor ELBL, and polypeptide film structures formed using ELBL will beuseful in a large number of different types of applications.Polypeptides designed using the method of the present invention havebeen shown to be useful for ELBL of film structures for possibleapplications in biomedical technology, food technology, andenvironmental technology. For example, such polypeptides could be usedto fabricate artificial red blood cells.

1. Artificial Red Blood Cells

A number of different approaches have been taken to red blood cellsubstitute development. One approach involves the use ofperfluorocarbons. Perfluorocarbon emulsions contain syntheticfluorinated hydrocarbons capable of binding oxygen and delivering it totissues. This approach however, increases reticulo-endothelial cellblockage. The perfluorocarbons can become trapped in thereticulo-endothelial system, which may result in adverse consequences.

Another approach focuses on antigen camouflaging, which involves coatingred blood cells with a biocompatible polymer called polyethylene glycol(PEG). The PEG molecules form permanent covalent bonds on the surface ofthe cell. The coating effectively hides the antigenic molecules on thesurface of the red blood cells, so that the blood recipient's antibodiesdo not recognize the cells as foreign. For example, the immune system ofa normal person who has type A blood will naturally have antibodies thatrecognize antigens on the surface of type B red blood cells, leading tocell destruction. The attachment of PEG to the surface of a type B redblood cell “camouflages” the surface of the cell, so that its surfaceantigens can no longer be recognized by the immune system and theantigenically-foreign red blood cells will not be destroyed as quickly(see Pargaonkar, N. A., G. Sharma, and K. K. Vistakula. (2001)“Artificial Blood: Current Research Report,” which is herebyincorporated by reference in its entirety).

A number of diseases, including thalassemia, that require repeated bloodtransfusions are often complicated by the development of antibodies to“minor” red cell antigens. This “allosensitization” can render thesepatients almost impossible to transfuse, rendering the situationlife-threatening. In in vitro testing, the PEG-modified red cells appearnot to trigger allosensitization and may also be useful in clinicalsituations where allosensitization has already occurred (see Scott, M.D. et al. (1997) “Chemical camouflage of antigenic determinants: Stealtherythrocytes,” Proc. Natl. Acad. Sci. USA. 94 (14): 7566-7571, which ishereby incorporated by reference in its entirety).

Other approaches involve purified hemoglobin. Unmodified cell-freehemoglobin has known limitations. These include oxygen affinity that istoo high for effective tissue oxygenation, a half-life within theintravascular space that is too short to be clinically useful, and atendency to undergo dissociation into dimers with resultant renaltubular damage and toxicity. Because of these limitations, hemoglobinused to make a cell-free red blood cell substitute must be modified. Anumber of modification techniques have been developed. Hemoglobin can becross-linked (a covalent bond between two molecules is made by chemicalmodification) and polymerized using reagents such as glutaraldehyde.Such modifications result in a product that has a higher P₅₀ (partialpressure of oxygen at which 50% of all oxygen-binding sites areoccupied) than that of normal hemoglobin, and an increase in the plasmahalf-life of up to 30 hours. The source of the hemoglobin for thispurpose can be human (outdated donated blood), bovine, or humanrecombinant. The solution of modified hemoglobin is prepared from highlypurified hemoglobin and taken through various biochemical processes, toeliminate phospholipids, endotoxins, and viral contaminants (see Nester,T. and Simpson, M (2000) “Transfusion medicine update,” BloodSubstitutes, which is hereby incorporated by reference in its entirety).Biopure Corporation (Cambridge, Mass.) has been using modifiedhemoglobin for their product, Hemopure.

The main potential adverse effect of modified hemoglobin solutions is anincrease in systemic and pulmonary vascular resistance that may lead toa decrease in cardiac index. Decreases in the cardiac index may impairoptimum oxygen delivery and outweigh the advantage of an oxygen-carryingsolution (see Kasper S. M. et al. (1998) “The effects of increased dosesof bovine hemoglobin on hemodynamics and oxygen transport in patientsundergoing preoperative hemodilution for elective abdominal aorticsurgery,” Anesth. Analg. 87: 284-91, which is hereby incorporated byreference in its entirety). One study has examined the utility of thesesolutions in the acute resuscitation phase of unstable trauma patients.Design of the study, however, was poor, and any role of the solutions ininfluencing ultimate patient outcome was unclear (see Koenigsberg D. etal. (1999) “The efficacy trial of diaspirin cross-linked hemoglobin inthe treatment of severe traumatic hemorrhagic shock,” Acad. Emerg. Med.6: 379-80, which is hereby incorporated by reference in its entirety).

Many of the problems of cell-free hemoglobin can be overcome byencapsulating it with an artificial membrane. Liposomes are being usedto encapsulate hemoglobin for use as a blood substitute. The approach istechnically challenging because not only must the hemoglobin beprepared, it must be encapsulated in relatively high concentration andyield. The final products must be sterile and the liposomes must berelatively uniform in size.

Encapsulated hemoglobin has several advantages over cell-freehemoglobin. Firstly, the artificial cell membrane protects hemoglobinfrom degradative and oxidative forces in the plasma. Secondly, themembrane protects the vascular endothelium from toxic effects ofhemoglobin. These relate to heme loss, the production O₂ free radicalsand vasoconstrictor effects of NO binding. Thirdly, encapsulationgreatly increases the circulating persistence of the hemoglobin.Moreover, encapsulated hemoglobin can be freeze-dried for convenientstorage.

Liposomal encapsulation involves phospholipids, as in cell membranes.One major problem associated with liposomal encapsulation, however, isthat it is very difficult to regulate the average size and distributionof liposomes. Another is that unlike red blood cells, liposomes areoften not very stable, as they ordinarily lack an organizedcytoskeleton. Yet another problem is that liposomes often consist ofmultiple layers of phospholipid. (A recent review of blood substitutedevelopment is presented in Stowell et al. (2001) Progress in thedevelopment of RBC substitutes, Transfusion 41:287-299, which is herebyincorporated by reference in its entirety. See also Chang, T. 1998“Modified hemoglobin-based blood substitutes: cross linked, recombinantand encapsulated hemoglobin,” Artificial Cell 74 Suppl 2:233-41, whichis hereby incorporated by reference in its entirety.)

Red blood cell substitutes employing polypeptides designed using themethod of the present invention should offer several advantages overapproaches to the development of red blood cell substitutes known in theart, including, without limitation, superior oxygen and carbon dioxidebinding functionality, lower production cost (large-scale and thereforelow-cost production is possible because bacteria can be used tomass-produce the peptides and because peptide ELBL can be automated),the possibility of using suitable preparations of hemoglobin as atemplate for ELBL, polypeptide biodegradability, the immune “inertness”of designed polypeptides based on blood protein structure, and thestructural stability exhibited by designed polypeptide films, whichexceeds that of liposomes. Polypeptide ELBL assembly yields semi-porousfilms, minimizing the amount of material required for fabricating ameans of encapsulation and enabling glucose, oxygen, carbon dioxide, andvarious metabolites to diffuse as freely through the films as a lipidbilayer. In contrast, other polymers potentially suitable for thispurpose have undesirable side effects—for example, polylactide degradesinto lactic acid, the substance that causes muscle cramps, and poly(styrene sulfonate) is not biocompatible.

Microcapsules could be formed of designed polypeptides to encapsulatehemoglobin to serve as a red blood cell substitute. Hemoglobinpolypeptide microcapsules could also be engineered to incorporateenzymes, including superoxide dismutase, catalase, and methemoglobinreductase, which are ordinarily important for red blood cell function.Moreover, the nanofabricated microcapsules can predictably bedehydrated, suggesting that artificial red blood cells made as describedherein could be dehydrated, without loss of function, particularlybecause hemoglobin can be lyophilized (i.e., freeze-dried) andreconstituted without loss of function, and polyion films are stable todehydration. This will be important for long-term storage, transport ofblood substitutes, battlefield applications (particularly in remotelocations), and space exploration.

Polypeptides designed using the method of the present invention couldalso be used for drug delivery.

2. Drug Delivery

Micron-sized “cores” of a suitable therapeutic material in “crystalline”form can be encapsulated by designed polypeptides, and the resultingmicrocapsules could be used for drug delivery. The core must beinsoluble under some conditions, for instance high pH or lowtemperature, and soluble under the conditions where controlled releasewill occur. The surface charge on the crystals can be determined byζ-potential measurements (used to determine the charge in electrostaticunits on colloidal particles in a liquid medium). The rate at whichmicrocapsule contents are released from the interior of the microcapsuleto the surrounding environment will depend on a number of factors,including the thickness of the encapsulating shell, the polypeptidesused in the shell, the presence of disulfide bonds, the extent ofcross-linking of peptides, temperature, ionic strength, and the methodused to assemble the peptides. Generally, the thicker the capsule, thelonger the release time—the principle resembles that of gel filtrationchromatography.

Some work has been done on sustained release from ELBL microcapsules(see Antipov, A., Sukhorukov, G. B., Donath, E., and Mohwald, H. (2001)J. Phys. Chem. B, 105:2281-2284 and Freemantle, M. (2002)Polyelectrolyte multilayers, Chem. Eng. News, 80: 44-48, both of whichare incorporated herein by reference in their entireties).Polyelectrolytes that have been used are PSS, PAH, PAA, PVS, PEI, andPDDA.

Polypeptides designed using the method of the present invention shouldoffer a number of advantages in the context of drug delivery, includingwithout limitation control over the physical, chemical, and biologicalcharacteristics of the microcapsule; the ability to make capsules with adiameter of less than 1 mm, making the capsules suitable for injection;low likelihood of eliciting an immune response; generally highbiocompatibility of capsules; control over the diffusive properties ofthe microcapsules by varying the thickness of the layers and usingcysteine, as discussed below; the ability to target specific locationsby modification of the microcapsule surface using the highly reactivesulfhydryl groups in cysteine (as is well known in the art, freesulfhydryl groups, free amino groups, and free carboxyl groups are sitesto which molecules for specific targeting could be attached), or byincorporation of a specific functional domain in the design of thepolypeptide; and the ability of microstructures to be taken up by cellsusing either endocytosis or pinocytosis.

Polypeptides designed using the method of the present invention couldalso be used for antimicrobial coatings.

3. Antimicrobial Coatings

The method of the present invention could be used to manufacture filmsencompassing antimicrobial peptides. For example, one suitable sequencemight be Histatin 5, which occurs in humans: Asp Ser His Ala Lys Arg HisHis Gly (SEQ ID NO: 8) Tyr Lys Arg Lys His Glu Lys His His Ser His ArgGly TyrThe preponderance of positive charge at slightly basic pH makes thissequence quite suitable for ELBL. It could be appended to a peptidedesigned using the method of the present invention, resulting in anantimicrobial peptide suitable for use in ELBL. This peptide could beused as an anti-biofouling coating. For instance, the peptide could beused to form a coating on devices used for implantation.

There are also a number of other areas in which polypeptides designedusing the method of the present invention could be useful.

4. Other Uses

Other possible uses for peptides designed using the method of thepresent invention include without limitation food covers, wraps, andseparation layers; food casings, pouches, bags, and labels; foodcoatings; food ingredient microcapsules; drug coatings, capsules, andmicrocapsules; disposable food service items (plates, cups, cutlery);trash bags; water-soluble bags for fertilizer and pesticides;microcapsules for fertilizer and pesticides; agricultural mulches; papercoatings; loose-fill packaging; disposable medical products (e.g. glovesand gowns); and disposable diapers.

C. Fabrication

Once amino acid sequence motifs have been selected from those identifiedusing the method discussed in Part VII(B)(1) above or designed de novo,the designed polypeptide is synthesized using methods well known in theart, such as solid phase synthesis and F-moc chemistry or heterologousexpression following gene cloning and transformation. Designedpolypeptides may be synthesized by a peptide synthesis company, forexample SynPep Corp. (Dublin, Calif.), produced in the laboratory usinga peptide synthesizer, or produced by recombinant methods.

In one embodiment, a designed polypeptide consists of individual aminoacid sequence motifs joined in tandem. The same motif may be repeated,or different motifs may be joined in designing a polypeptide for ELBL.Moreover, functional domains may be included, as discussed above. Otheramino acids than glycine could be used to link the sequence motifs, andamino acids other than the 20 usual ones could be included in the motifsthemselves, depending on the properties desired of the polypeptide.Other properties could likewise be specified by design requirements,using methods known in the art. For example, proline could be includedfor turn formation, glycine for chain flexibility, and histidine forpH-sensitive charge properties near neutral pH. “Hydrophobic” aminoacids could also be included—hydrophobic residue content could play arole in assembly behavior and contribute to layer stability in a wayresembling the hydrophobic stabilization of globular proteins.

It is preferred that fabricated polypeptides be at least 15 amino acidslong, although it is more preferred that the fabricated polypeptides beat least 32 amino acids long. The reason for this is that the entropyloss per molecule is so thermodynamically unfavorable for short polymersthat adsorption to an oppositely-charged surface is inhibited, even ifthe polypeptide has a charge per unit length of 1; long polyelectrolytesadsorb better than short ones. This is illustrated in FIG. 12. Theaverage molecule masses of the peptides utilized for thelength-dependence studies were 1,500-3,000 Da (poly-L-glutamate,“small”), 3,800 Da (poly-L-lysine, “small”), 17,000 Da(poly-L-glutamate, “medium”), 48,100 Da (poly-L-lysine, “medium”),50,300 Da (poly-L-glutamate, “large”), and 222,400 Da (poly-L-lysine,“large”). The data shown in FIG. 12 clearly indicate that ELBL dependson length of peptide. Inclusion of Cys enables the use of relativelysmall peptides for ELBL, because the sulfhydryl group can be used toform disulfide crosslinks between polypeptides.

D. Experiments

1. Example 1 Design of Polypeptides Based on Human Blood ProteinSequences and Their Use in Polypeptide Film Fabrication

For this work, amino acid sequences were selected using the processdescribed in Part VII(B)(1) above to identify sequence motifs in theprimary structure of human blood proteins: Complement C3 (gi|68766) wasthe source of the anionic sequence motifs, and lactotransferrin(gi|4505043) the source of the cationic sequence motifs. As discussedabove, blood protein sequences were used to minimize the immune responseof patients into whom devices involving the polypeptides might beintroduced (including, e.g. artificial red blood cells). In principle,this approach should be applicable for any organism having an immunesystem; it is not limited to humans. Polypeptides were synthesized bySynPep Corp. (Dublin, Calif.). The polypeptide sequences were: Tyr GluGlu Asp Glu Cys Gln Asp Gly (SEQ ID NO: 2) Glu Glu Asp Glu Cys Gln AspGly Glu Glu Asp Glu Cys Gln Asp Gly Glu Glu Asp Glu Cys Gln Asp Tyr ArgArg Arg Arg Ser Val Gln Gly (SEQ ID NO: 1) Arg Arg Arg Arg Ser Val GlnGly Arg Arg Arg Arg Ser Val Gln Gly Arg Arg Arg Arg Ser Val Gln Tyr GluGlu Asp Glu Cys Gln Asp Gly (SEQ ID NO: 4) Glu Glu Asp Glu Cys Gln AspGly Glu Glu Asp Glu Cys Gln Asp Gly Glu Glu Asp Glu Cys Gln Asp Gly GluGlu Asp Glu Cys Gln Asp Gly Glu Glu Asp Glu Cys Gln Asp Tyr Arg Arg ArgArg Ser Val Gln Gly (SEQ ID NO: 3) Arg Arg Arg Arg Ser Val Gln Gly ArgArg Arg Arg Ser Val Gln Gly Arg Arg Arg Arg Ser Val Gln Gly Arg Arg ArgArg Ser Val Gln Gly Arg Arg Arg Arg Ser Val Gln

The amino acid residues are represented by the three-letter code givenabove. One glycine was introduced between each 7-residue motif toinhibit secondary structure formation. Glycine was selected for thispurpose because it allows the greatest variability in combination ofdihedral angles (see Ramachandran, G. N. and Saisekharan, V. (1968),Adv. Protein Chemistry, 23:283, which is incorporated by referenceherein in its entirety) and has a low helix propensity (0.677) and lowsheet propensity (0.766). Alternatively, proline could be substitutedfor glycine between motifs on the basis of calculated structurepropensities. Additionally, a single tyrosine was included at theN-terminus of each peptide for concentration determination by UVabsorption at 280 nm.

The polypeptides were named SN1 (SEQ ID NO: 2), SP2 (SEQ ID NO: 1), LN3(SEQ ID NO: 4), and LP4 (SEQ ID NO: 3), respectively, meaning shortnegative, short positive, long negative, and long positive. Thesesequences are quite different from polylysine (commonly used in the artas a polycation) and polyglutamate (commonly used in the art as apolyanion) which, though available commercially and inexpensive, have ahigh α-helix propensity under conditions of mild pH and, crucially, areimmunoreactive. The calculated charge per unit length on the designedpeptides at neutral pH is 0.5 electrostatic units for SP and LP and 0.6electrostatic units for SN and LN. The positive peptides are somewhatmore hydrophobic than the negative ones, owing to the presence of valineand the long hydrocarbon side chain of arginine. (As mentioned above,hydrophobic interactions between polypeptide layers could stabilizefilms to some extent.) The lengths are consistent with published studiesshowing that polyions must have greater than 20 charged groups (i.e.aspartic acid and glutamic acid; lysine, arginine, and histidine) to besuitable for ELBL (see Kabanov, V. and Zezin, A. (1984) Pure Appl. Chem.56:343 and Kabanov, V. (1994) Polym. Sci. 36:143, both of which areincorporated by reference herein in their entireties).

a. Experimental Demonstration

i. Materials

QCM electrodes (USI-System, Japan) coated with evaporated silver had asurface area of 0.16±0.01 cm² on each side, a resonant frequency of 9MHz (AT-cut), and a long-term stability of ±2 Hz. The polypeptidemolecular weight was verified by electrospray mass spectrometry. Peptidepurity was greater than 70%. The polypeptide buffer was 10 mM sodiumphosphate or 10 mM Tris-HCl, 1 mM DTT, 0.1 mM sodium azide, pH 7.4. Allchemicals other than polypeptides were purchased from Sigma-Aldrich(USA).

ii. Procedures

Experiments were done using pairs of designed polypeptides, one negativeand one positive. Multilayer films consisting of at least 5 bi-layers ofthe above-identified SP2, SN1, LP4, and LN3 were deposited onto the QCMresonators using standard ELBL techniques (a bi-layer consists of onelayer of polycation and one layer of polyanion). The polypeptideconcentration used for layer adsorption was 2 mg-mL⁻¹. It is known thatdependence of polyion layer thickness on polyelectrolyte concentrationis not strong (see Lvov, Y. and Decher, G. (1994) Crystallog. Rep.39:628, which is incorporated herein by reference in its entirety); inthe concentration range 0.1 to 5 mg mL⁻¹, bilayer thickness wasapproximately independent of concentration for PSS/PAH. By contrast,polypeptide thin films appear substantially less thick than thosefabricated using high molecular weight PSS/PAH (mass calculated using Δfdata using the well-known Sauerbrey equation); see Lvov, Y. and Decher,G. (1994) Crystallog. Rep. 39:628. This follows from calculating filmthickness on the basis of mass deposited, as is ordinarily done in theart for proteins. The calculated thickness for the designed polypeptideassembly shown in FIG. 3(c) is greater than the end-to-end length of thepeptides used to make the film. DTT was included at 1 mM to inhibitdisulfide bond formation. The adsorption time was 20 minutes.

Resonators were rinsed for 1 min. in pure water between subsequentadsorption cycles (removing perhaps 10-15% of weakly adsorbed material)and dried in a stream of gaseous N₂. Then the mass of the depositedpeptide was measured indirectly by QCM. The mass measurement includessome water, despite drying, and low mass ions like K⁺, Na⁺, and Cl⁻.Partial interpenetration of neighboring layers of peptide is probable(see Decher, G. (1997) Science 227:1232; Schmitt et al. (1993)Macromolecules 26:7058; and Komeev et al. (1995) Physica B 214:954);this could be important for the efficiency of disulfide “locking.”

iii. Results

After adsorption of the polypeptide and rinsing and drying the QCMresonator, the resonant frequency of the resonator was measured. Thisenabled calculation of the frequency shift on adsorption and change inadsorbed mass. A decrease in frequency indicates an increase in adsorbedmass. The results are provided in FIGS. 3(a) and 3(b). FIG. 3(a) shows acomparison of adsorption data for LP4 and LN3 in different buffers (10mM sodium phosphate, pH 7.4, 1 mM DTT and 10 mM Tris-HCl, pH 7.4, 1 mMDTT). It is clear from these data that adsorption depends more on theproperties of the peptides than the specific properties of the bufferused. FIG. 3(b) shows resonator frequency versus adsorbed layer fordifferent combinations of SP2, SN1, LP4, and LN3 (namely, SP2/SN1,SP2/LN3, LP4/SN1, and LP4/LN3) in 10 mM sodium phosphate, pH 7.4 and 1mM DTT (the lines merely connect experimental data points). Each ofthese combinations involved one negative polypeptide and one positivepolypeptide, as required by ELBL. FIG. 3(c) shows a graph of calculatedadsorbed mass versus layer number for SN1 and LP4 in 10 mM Tris-HCl, pH7.4 and 1 mM DTT (calculated from experimental data using the Sauerbreyequation). The total adsorbed mass, approximately 5 μg, correspondsapproximately to 1 mmol of peptide. The equation used for thiscalculation was Δm=−0.87·10⁻⁹ Δf, where m is mass in grams and f isfrequency in Hz (see Lvov, Y., Ariga, K., Ichinose, I., and Kunitake, T.(1995) J. Am. Chem. Soc. 117:6117 and Sauerbrey, G. (1959) Z. Physik155:206, both of which are incorporated herein by reference in theirentireties). Film thickness, d, is estimated as d=−0.016Δf where d is innm (see Yuri Lvov, “Electrostatic Layer-by-Layer Assembly of Proteinsand Polyions” in Protein Architecture: Interfacial Molecular Assemblyand Immobilization Biotechnology, (Y. Lvov & H. Möhwald eds., 2000) (NewYork: Dekker, 2000) pp. 125-167, which is incorporated herein byreference). The line in FIG. 3(c) is a linear fit to experimental datapoints. The linearity of the data is a likely indicator of precise,regular assembly during adsorption and an approximately uniform densityof the polypeptides in each adsorbed layer. Adsorption occurred with afrequency shift of −610±60 Hz (cations) or −380±40 Hz (anions). Lineargrowth of deposited polypeptide mass indicates repeatability ofadsorption steps early in the assembly process and the general successof the multilayer fabrication process.

iv. Conclusions

The above results show that polypeptides designed using the method ofthe present invention are suitable for ELBL, despite significantqualitative differences from PSS and PAH, flexible homopolymers having 1charge per unit length at pH 7.4. The charge per unit length onpoly-L-lysine and poly-L-glutamic acid is 1 at pH 7.4, as with PSS andPAH, but both of these polypeptides have a marked propensity to formα-helical structure under various conditions, making them substantiallyless suitable for multilayer assembly when control over thin filmstructure is desired. The monodisperse polypeptides of the presentinvention, however, enable the practitioner to know, quite precisely,the structure of the material being used for ELBL. Moreover, usualcommercial preparations of poly-L-lysine and poly-L-glutamic acid arepolydisperse, and poly-L-lysine, poly-L-glutamic acid, PSS, and PAHevoke an immune response (i.e. are immunogenic) in humans.

Because the designed polypeptides are readily adsorbed on an oppositelycharged surface, as demonstrated by experiment, there is no need for a“precursor” layer. As is known in the art, “precursor” layers aredeposited on a substrate to enhance adsorption of less adsorptivesubstances. The lack of a precursor layer enhances the biocompatibilityof the polyion films because polymers ordinarily used as precursors areimmunogenic or allow less precise control over polymer structure or thinfilm structure than designed polypeptides.

Multilayers of the designed polypeptides were stable at the pH of humanblood, 7.4. Thus, the multilayers should be useful for a broad range ofbiological applications. Adsorption of the designed polypeptides, eachof less than 1 charge per residue, was essentially complete in less than10 min. at 2 mg/mL and low ionic strength. This implies that thesepolypeptides can be used to form multilayer films quickly and withrelative ease. Drying the peptide film with N_(2(g)) after deposition ofeach layer did not impair assembly. Drying is done to get an accurateQCM frequency measurement, but is not required for assembly.

The film assembly experiments were done at a lower ionic strength thanthat of blood, but the process gives a qualitatively similar result athigher ionic strength. The chief difference is the amount of peptidedeposited per adsorption layer—the higher the ionic strength, thegreater the amount of peptide deposited. This is illustrated by thegraph in FIG. 7, which shows the amount of material deposited as afunction of ionic strength—the peptides used were poly-L-glutamic acidand poly-L-lysine. QCM resonant frequency is plotted against adsorptionlayer. The average molecular mass of poly-L-glutamate was 84,600 Da,while that of poly-lys was 84,000 Da. The peptide concentration used forassembly was 2 mg/mL. The data indicate salt concentration (ionicstrength of solution) influences thin film assembly. In general, theamount of material deposited per layer increases with ionic strength inthe range 0-100 mM NaCl. As the essential character of ELBL withdesigned polypeptides appears not to depend on the choice of bufferunder conditions of relatively high net charge per unit length and lowionic strength, qualitatively similar results are expected at the ionicstrength of human blood. Thus, the choice of buffer should notfundamentally alter the stability of the multilayers in their targetenvironment. However, even if the choice of buffer did affect thestability of the multilayers, the “locking” mechanism would be availableas a design feature to stabilize the capsule.

The greater apparent deposition of positive polypeptides than negativeones may result from the higher charge per unit length on the positivepolypeptides at pH 7.4. The material deposited in each layer probablycorresponds to that required for neutralization of the charge of theunderlying surface. Hydrophobic interactions could also help to explainthis feature of adsorption behavior.

The usual thin film thickness calculation for proteins and otherpolymers is probably invalid for short polypeptides (calculatedthickness is 60-90 nm, but summed length of 10 polypeptides isapproximately 120 nm). This probably results from a high density ofpacking of the relatively short polypeptides onto the adsorptionsurface; the result is also consistent with finding that film thicknessvaries with ionic strength, as changes in structural properties of apolymer will occur and screening of charges by ions will reduceintra-layer charge repulsion between adsorbed peptides. The thickness ofthe designed polypeptide thin film discussed here is estimated at 20-50nm.

Many aspects of the design and fabrication cycles could be automated.For example, a computer algorithm could be used to optimize the primarystructure of peptides for ELBL by comparing predicted peptide propertieswith observed physical properties, including structure in solution,adsorption behavior, and film stability at extremes of pH. Moreover, thepolypeptide film assembly process can be mechanized, once the details ofthe various steps have been sufficiently determined.

2. Example 2 Experiments Involving De Novo-Designed PolypeptidesContaining Cysteine

a. Polypeptides

The polypeptides used were: Tyr Lys Cys Lys Gly Lys Val Lys Val (SEQ IDNO: 5) Lys Cys Lys Gly Lys Val Lys Val Lys Cys Lys Gly Lys Val Lys ValLys Cys Lys Gly Lys Val Lys Tyr Glu Cys Glu Gly Glu Val Glu Val (SEQ IDNO: 6) Glu Cys Glu Gly Glu Val Glu Val Glu Cys Glu Gly Glu Val Glu ValGlu Cys Glu Gly Glu Val GluUnlike the other polypeptides used in the experiments described herein,these two were not designed using human genome information; they weredesigned for the sole purpose of assessing the role of disulfide bondformation in polypeptide film stabilization.

b. Procedures

All experiments were conducted at ambient temperature.

All assembly experiments using QCM were conducted in the sameconditions, except that the samples to undergo oxidation were driedusing air instead of nitrogen gas. The assembly conditions were 10 mMTris-HCl, 10 mM DTT, pH 7.4. The nominal peptide concentration was 2mg/ml. The number of layers formed was 14.

Disulfide locking conditions for the oxidizing samples were 10 mMTris-HCl, 1% DMSO, saturation of water with air, pH 7.5. The duration ofthe “locking” step was 6 hours. Conditions for the reducing samples were10 mM Tris-HCl, 1 mM DTT, saturation of water with nitrogen, pH 7.5. Theduration of this step was 6 hours.

All disassembly experiments using QCM were conducted in the sameconditions, except that the oxidizing samples were dried using airinstead of nitrogen. Disassembly conditions were 10 mM KCl, pH 2.0Samples were rinsed with D.I. water for 30 seconds prior to drying.

Three different types of experiments were conducted: (1) Reducing—notreatment: disassembly was conducted immediately after assembly; (2)Reducing—6 hours, as described above for reducing samples; and (3)Oxidizing—6 hours, as described above for oxidizing samples.

c. Results

The results are illustrated in FIG. 10. In the first two experiments(both reducing), all of the deposited material (100%) disassembledwithin 50 minutes. By contrast, in the oxidizing experiment, asubstantial amount of material remained after substantial incubation ofthe peptide film-coated QCM resonator at pH 2 for over 5 hours. Thestability of the polypeptide films at acidic pH is determined by theconditions of assembly; in this way, film or capsule stability is adesign feature that becomes possible by using polypeptides as thepolyelectrolytes for ELBL.

d. Conclusions

Electrostatic forces play a key role in holding togetheroppositely-charged layers of designed polypeptides. At acidic pH, thenet charge on one of the peptides is neutralized and the polypeptidefilm disassembles due to electrostatic repulsion. Reducing conditionsprevent disulfide bond formation. Therefore, the electrostaticattraction between the layers is the only binding force for stabilizingthe layers under these conditions. By contrast, under oxidizingconditions disulfide bonds are formed. At acidic pH, disulfide bondsinhibit film disassembly. The results indicate that layer stability atacidic pH is directly affected by the formation of intra- and/orinter-layer disulfide bonds—i.e. between molecules in the same layer,between molecules in adjacent layers, or both. This is illustrated bythe results shown in FIG. 10—due to disulfide locking, more than 30% ofthe film remained stable at acidic pH, despite electrostatic repulsionat relatively low ionic strength. Peptides with more cysteine residuesare anticipated to further improve disulfide locking efficiency.Moreover, adjustment of the conditions of peptide assembly will be animportant aspect of engineering films to have the desired physical aswell as chemical and biological properties.

3. Example 3

Experiments Involving Designed Polypeptides Containing Cysteine

a. Materials

The essential elements of this experiment were a quartz crystalmicrobalance instrument; silver-coated resonators (9 MHz resonantfrequency); the negative 48-residue peptide (LN3) (SEQ ID NO: 4); and apositive 48-residue peptide named “SP5” of the following sequence: TyrLys Gly Lys Lys Ser Cys His Gly (SEQ ID NO: 7) Lys Gly Lys Lys Ser CysHis Gly Lys Gly Lys Lys Ser Cys His Gly Lys Gly Lys Lys Ser Cys His

Like the other designed peptides discussed above in Part VII(E)(1), SP5was designed using the process described above in Part VII(B)(1) toanalyze the amino acid sequence of the human blood proteinlactotransferrin (gi|4505043). The ELBL buffer was 10 mM Tris, pH 7.4,10 mM NaCl, and 1 mM DTT. The disassembly buffer was 10 mM KCl, pH 2. 2mL peptide solutions were prepared for SP5 and LN3 by adding 4 mg ofeach peptide to 2 mL of the above buffer solution and adjusting the pHof each solution to 7.4; the peptide concentration was 2 mg-mL⁻¹.

b. Procedure for Monitoring Assembly of Polypeptide Layers on QCMResonators

Reducing procedures were as follows: (1) The frequency of the resonatorwas measured and recorded prior to peptide adsorption; (2) The resonatorwas dipped into the SP5 peptide solution for 20 min.; (3) The resonatorwas dipped into the SP5 rinse solution for 30 sec.; (4) The resonatorwas removed from the rinse solution and dried using nitrogen gas; (5)The QCM resonant frequency of the resonator was recorded; (6) Theresonator was dipped into the LN3 peptide solution for 20 min.; (7) Theresonator was dipped into the LN3 rinse solution for 30 sec.; (8) Theresonator 1 was removed from the rinse solution and dried using nitrogengas; (9) The QCM resonant frequency of the resonator was recorded; (10)Steps 2 through 9 were repeated until 16 layers were adsorbed onto theresonator.

Oxidizing procedures were the same as the reducing procedures, exceptthat the resonator was rinsed in D.I. water instead of the SP5 buffer orthe LN3 buffer and dried with air instead of nitrogen before eachmeasurement.

c. Locking Procedures

Reducing procedures were as follows: The resonator was placed in anaqueous solution containing 1 mM DTT for 6 hours. DTT, a reducing agent,inhibited disulfide bond formation.

Oxidizing procedures were as follows: The resonator was placed in anair-saturated aqueous solution containing 1% DMSO for 6 hours. DMSO, anoxidizing agent, promoted disulfide bond formation.

d. Disassembly on Resonator

i. Solutions

Reducing conditions were as follows: 10 mM KCl, 1 mM DTT, pH 2.

Oxidizing conditions were as follows: 10 mM KCl, 20% DMSO, pH 2.

ii. Procedure for Disassembly

Reducing procedures were as follows: (1) The initial resonant frequencyof the resonator was measured by QCM and recorded; (2) The resonator wasdipped into the reducing disassembly solution for 5 min.; (3) Theresonator was rinsed in reducing buffer solution for 30 sec.; (4) Theresonator was dried with gaseous N₂; (5) The resonant frequency of theresonator was measured by QCM and recorded; (6) Steps 2 through 5 wererepeated for reading times of 5, 10, 15, 20, 30, 60, and 90 min.

Oxidizing procedures were the same as for reducing procedures, exceptthat rinsing of the resonator was done in D.I. water saturated with airinstead of reducing buffer.

e. Results

FIG. 8 shows approximately linear increase in mass deposited during thinfilm assembly of SP5 and LN3. Both resonators show almost identicaldeposition of mass throughout the process of assembly, despitedifferences in assembly conditions.

FIG. 9 shows the percentage of material remaining during filmdisassembly. The layers subjected to oxidizing conditions showed aminimal loss of material at acidic pH with almost 90 to 95% of massretention. By contrast, layers subjected to reducing conditions lostalmost all the film material within the first 5 minutes of exposure toacidic pH.

f. Conclusions

The results demonstrate that at acidic pH, disulfide bonds prevent layerdegeneration and hold the layers firmly together. Layer stability atacidic pH is directly affected by the formation of intra- and/orinter-layer disulfide bonds. Disulfide bond formation is dependent onthe concentration and proximity of cysteine residues to each other.Increasing the concentration per unit chain length of the polypeptidewould therefore directly influence disulfide bond formation and thinfilm stability. Increasing the ionic strength of the buffer solutionsused for film assembly influences the concentration of cysteine in thefilm by increasing the amount of material deposited per adsorption cycleand the thickness of each layer. The increased number of cysteine aminoacids in a single layer would in this way increase the number ofdisulfide bonds formed, and, on oxidation, increase film stability.

Other embodiments of the invention are possible and modifications may bemade without departing from the spirit and scope of the invention.Therefore, the detailed description above is not meant to limit theinvention. Rather, the scope of the invention is defined by the appendedclaims.

1. An artificial red blood cell, comprising: a thin film encapsulating acore, the core comprising hemoglobin, wherein the film comprises aplurality of layers of polypeptides, the layers comprising alternatingoppositely charged polypeptides, wherein a first layer comprises a firstlayer polypeptide comprising one or more first amino acid sequencemotifs, wherein the one or more amino acid sequence motifs consist of namino acids, at least x of which are positively charged, or at least xof which are negatively charged, wherein x is greater than or equal toapproximately one-half of n.
 2. The artificial red blood cell of claim1, wherein the amino acid sequence motif is found in the primarystructure of a human blood protein.
 3. The artificial red blood cell ofclaim 2, wherein the human blood protein is complement C3, orlactotransferrin.
 4. The artificial red blood cell of claim 1, whereinthe hemoglobin is in liquid form or in crystallized form.
 5. Theartificial red blood cell of claim 1, wherein n is 4 to
 12. 6. Theartificial red blood cell of claim 1, wherein the first amino acidsequence motif comprises a cysteine residue.
 7. The artificial red bloodcell of claim 1, comprising a second layer disposed on the first layer,the second layer comprising a second layer polypeptide comprising one ormore second amino acid sequence motifs having a charge opposite that ofthe amino acid sequence motif of the first layer polypeptide.
 8. Theartificial red blood cell of claim 1, further comprising a superoxidedismutase, a catalyse, a methemoglobin reductase, or a combinationcomprising one or more of the foregoing molecules.
 9. The artificial redblood cell of claim 1, wherein the first layer polypeptide isbiocompatible.
 10. An artificial red blood cell, comprising: a thin filmencapsulating a core, the core comprising hemoglobin, wherein the filmcomprises a plurality of layers of polypeptides, opposing layerscomprising alternating oppositely charged polypeptides, and wherein atleast half of the amino acid residues in the oppositely chargedpolypeptides have the same charge.
 11. The artificial red blood cell ofclaim 10, wherein the film comprises a first layer polypeptidecomprising one or more first amino acid sequence motifs, wherein the oneor more amino acid sequence motifs consist of n amino acids, at least xof which are positively charged, or at least x of which are negativelycharged, wherein x is greater than or equal to approximately one-half ofn.
 12. The artificial red blood cell of claim 10, wherein the filmcomprises a second layer polypeptide having a charge opposite the chargeof the first layer polypeptide and comprising one or more second aminoacid sequence motifs, wherein the one or more amino acid sequence motifscomprises consist of n amino acids, at least x of which are positivelycharged, or at least x of which are negatively charged, wherein x isgreater than or equal to approximately one-half of n.
 13. The artificialred blood cell of claim 10, wherein the first amino acid sequence motifcomprises a cysteine residue.
 14. The artificial red blood cell of claim10, wherein the artificial red blood cell further comprises a layerwhich is not a polypeptide.